Interferon gamma (IFNgamma) has been implicated in the pathogenesis of autoimmune (Hashimoto's) thyroiditis. We have generated transgenic mice that express IFNg specifically in the thyroid gland, and shown that IFNgamma a) directly induces primary hypothyroidism with decreased NIS expression; b) induces Hurthle cells-like and increase thyroidal fat; c) protects from autoimmune thyroiditis. Specific aim 1 is to test the hypothesis that the IFNg-driven activation of the immunoproteasome is critical to the ontogeny of Hurthle cells. We will: i) test whether pharmacologic or genetic blocks of the immunoproteasome inhibit the appearance of Hurthle cells; ii) evaluate in situ immunoproteasome expression in thyroids from thyr-IFNgamma transgenic mice, as well as from patients with Hurthle cell lesions. Specific aim 2 is to unravel the mechanism through which IFNgamma protects from autoimmune thyroiditis. We hypothesize that this protective effect is mediated by a caspase-8 dependent activation of apoptosis, which results in death of autoreactive, thyroid-specific lymphocytes. We will transduce autoreactive T cells with an inactive form of caspase-8 and adoptively transfer T cells to wild type or thyr-IFNgamma transgenic hosts, assessing thereafter the incidence and severity of thyroiditis. We wil also cross thyr-IFNgamma transgenics to knockout mice that lack IRF-1 or IRF-4, two critical transcription factors linking IFNgamma signaling with caspase-8 dependent apoptosis. Specific aim 3 is to study the interaction between IFNg interleukin-12 (IL-12) on thyroid autoimmunity and function. We have generated transgenic mice that express IL-12 specifically in the thyroid gland. The mice develop spontaneously lymphocytic thyroiditis and primary hypothyroidism with increased NIS expression. We will cross thyr-IL-12 transgenics to thyr-IFNg transgenics to assess whether the disease suppressive effect of IFNgamma prevails on the disease-promoting effect of IL-12, and whether IFNg and IL-12 synergistically induce a more severe form of hypothyroidism. Specific aim 4 is to investigate why thyroids from thyr-IFNg transgenic contain an increased number of adipocytes, and to assess whether this increase influences thyroid function. We hypothesize that thyroid macrophages primed in vivo by IFNg promote differentiation of stromal preadipocytes into mature adipocytes. We will co-culture thyroid macrophages from transgenics and controls with syngeneic pre-adipocytes, monitoring acquisition of morphological features typical of adipocytes. We also hypothesize that augmented TSH signaling is required to induce the adipocyte-rich phenotype, and cross thyr-IFNg transgenics to TSH receptor knockout mice.